Compound feed for improving disease resistance of breeding ducks and preparation process thereof

Abstract

The application provides a compound feed for improving the disease resistance of breeding ducks and a preparation process thereof, and belongs to the technical field of breeding feeds; the preparation process comprises the following steps: preparing modified resistant starch acetate; preparing a bifidobacterium composite microbial agent; preparing a composite nano carrier; preparing an extract composite additive; and preparing a compound feed; after the resistant starch acetate is activated by 1,1-carbonyl diimidazole and then a concanavalin A solution is added for reaction, the modified resistant starch acetate is obtained; after the bifidobacterium is loaded by using the modified resistant starch acetate, the bifidobacterium can be protected from damage by gastric acid and digestive enzymes, and then the number of bifidobacterium reaching the large intestine of the breeding duck is improved, which is beneficial to better inhibiting the reproduction of harmful bacteria in the large intestine of the breeding duck and improving the disease resistance of the breeding duck; in addition, after the resistant starch acetate is modified by using the concanavalin A, the loading rate of the resistant starch acetate to the bifidobacterium is improved.

Description

Technical Field

[0001] This invention relates to the field of aquaculture feed technology, specifically to a compound feed for improving the disease resistance of breeding ducks and its preparation process. Background Technology

[0002] In current duck farming, laying ducks are often kept in open or semi-open duck houses, making it difficult to effectively control hygiene. The damp environment inside the duck houses provides a breeding ground for bacteria and viruses, leading to viral diseases such as duck plague and avian influenza, as well as bacterial infections such as E. coli, which are the main health threats. In the past, the method of adding antibiotics to feed to prevent and treat diseases not only caused pathogens to develop drug resistance, resulting in a gradual decrease in the therapeutic effect of antibiotics, but also caused drug residue problems.

[0003] Bifidobacteria, as a dominant probiotic in the gut, can reduce the risk of intestinal infection, promote the proliferation and differentiation of lymphocytes, enhance the phagocytic capacity of macrophages, and strengthen humoral and cellular immunity. However, when Bifidobacteria are added directly to feed, laying ducks have high gastric acid secretion. After Bifidobacteria enter the stomach orally, the strong acid environment will cause the bacterial protein to denature, the cell membrane to rupture, and the number of live bacteria to drop significantly.

[0004] Resistant starch is a type of carbohydrate that is not easily hydrolyzed by amylases in the digestive tract. Through physical encapsulation, structural barriers, and microenvironment regulation, it can significantly improve the survival rate of Bifidobacteria in the acidic environment of the stomach. However, its loading of Bifidobacteria usually relies on van der Waals forces, electrostatic interactions, or hydrogen bonds, resulting in weak binding forces. Moreover, natural resistant starch has high crystallinity and a tight molecular arrangement, with internal pore diameters smaller than the size of Bifidobacteria. Bacteria cannot enter the carrier and can only be adsorbed on the surface, leading to a low loading rate of Bifidobacteria and limited effect on enhancing the immunity of breeding ducks.

[0005] Therefore, there is a need to develop a compound feed that can enhance immunity and improve the disease resistance of breeding ducks, as well as its preparation process. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a compound feed for improving the disease resistance of breeding ducks and its preparation process.

[0007] A process for preparing a compound feed to improve the disease resistance of breeding ducks includes the following steps:

[0008] S1: Preparation of modified resistant starch acetate

[0009] First, resistant starch acetate was prepared using resistant starch and acetic anhydride as raw materials. After activation, it was reacted with concanavalin A to produce modified resistant starch acetate.

[0010] S2: Preparation of Bifidobacterium compound inoculum

[0011] After activating the lyophilized Bifidobacterium powder, a Bifidobacterium suspension was prepared, which was then mixed with a modified resistant starch acetate solution. This mixture was then added to a mixture of Span 80 and liquid paraffin, homogenized and emulsified, and then cross-linked with calcium chloride solution to prepare a Bifidobacterium compound inoculum.

[0012] S3: Preparation of composite nanocarriers

[0013] Bacterial cellulose was acidified, dispersed in PBS buffer, and then mixed with activated oleic acid to react and prepare a composite nanocarrier.

[0014] S4: Preparation of extract composite additives

[0015] After dissolving the above-mentioned composite nanocarrier, it was mixed with verbena extract solution and moringa leaf extract solution, and then added to a chloroform solution of Tween 80. The mixture was homogenized and emulsified to prepare an extract composite additive.

[0016] S5: Preparation of compound feed

[0017] After mixing and crushing corn, broken rice, soybean meal and eggshell powder, add the above-mentioned extract compound additive, bifidobacteria compound inoculant, xylooligosaccharide, compound microorganisms and spirulina powder, mix thoroughly and granulate to obtain compound feed.

[0018] Furthermore, step S1 specifically includes the following steps:

[0019] S1.1: Add resistant starch to deionized water at a ratio of 1g:(10-20)mL, stir thoroughly to form a suspension, and cool in an ice bath to 3-5℃ to obtain a resistant starch suspension;

[0020] S1.2: Add 0.1 mol / L sodium hydroxide solution to the above resistant starch suspension to adjust the pH to 8-10, and stir to swell for 20-30 min. Then add acetic anhydride, stir and react at 20-30℃ for 2-3 h, add 0.1 mol / L hydrochloric acid solution to adjust the pH to neutral, collect the precipitate by centrifugation, wash and vacuum dry to obtain resistant starch acetate.

[0021] S1.3: The above resistant starch acetate was suspended in acetone at a ratio of 1 g: (20-30) mL, and 1,1-carbonyldiimidazole was added. The mixture was stirred and activated at 25-35 °C for 10-12 h to obtain an activated resistant starch acetate solution, wherein the mass ratio of 1,1-carbonyldiimidazole to resistant starch acetate was 1: (4-5).

[0022] S1.4: Dissolve canavalia protein A in PBS buffer at a ratio of 1g:(150-200)mL, then add it to the above activated resistant starch acetate solution, stir at 3-5℃ for 12-16h, then dialysis for purification and freeze-drying to obtain modified resistant starch acetate.

[0023] Furthermore, S2 specifically includes the following steps:

[0024] S2.1: Inoculate the lyophilized Bifidobacterium powder into MRS medium, anaerobically culture at 37℃ for 40-48 h, centrifuge at 4℃, discard the supernatant, wash 2-3 times with sterile physiological saline, and then resuspend in sterile PBS buffer to obtain a concentration of 10. 10 Bifidobacterium suspension with CUF / mL;

[0025] S2.2: Dissolve the modified resistant starch acetate obtained in step S1.4 in sterile PBS buffer at a ratio of 1 g: (20-30) mL, then add the above-mentioned Bifidobacterium suspension, stir and mix evenly to obtain an aqueous phase, wherein the concentration of Bifidobacterium in the aqueous phase is (4-5) × 10⁻⁶. 9 CUF / mL;

[0026] S2.3: Add Span 80 to liquid paraffin at a ratio of 1g:(40-50)mL, stir to dissolve, add the above aqueous phase, and homogenize and emulsify at 1500-2500rpm for 10-20min to obtain an emulsion, wherein the volume ratio of aqueous phase to liquid paraffin is 1:2.

[0027] S2.4: Add 0.1 mol / L calcium chloride solution to the above emulsion, heat and stir at 37°C for 20-30 min for cross-linking, then centrifuge at 3-5°C, discard the supernatant, wash with sterile petroleum ether and sterile PBS buffer, and freeze dry to obtain Bifidobacterium complex agent, wherein the volume ratio of calcium chloride solution to water phase is 1:(6-8).

[0028] Furthermore, S3 specifically includes the following steps:

[0029] S3.1: Add bacterial cellulose to phosphate buffer solution with pH 5-6 at a ratio of 1g:(20-30)mL, and sonicate for 30-40min to obtain bacterial cellulose suspension;

[0030] S3.2: Add a 64% sulfuric acid solution to the above bacterial cellulose suspension, stir and hydrolyze at 40-50℃ for 14-16h, then add 1mol / L sodium hydroxide solution to adjust the pH to neutral, and obtain acidified bacterial cellulose by centrifugation, washing and freeze drying. The volume ratio of sulfuric acid solution to bacterial cellulose suspension is 1:(2-3).

[0031] S3.3: Add oleic acid to chloroform at a volume ratio of 1:(8-10) mL, stir and mix thoroughly, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir and activate in the dark for 1-2 h to obtain an activated oleic acid solution.

[0032] S3.4: Disperse the above acidified bacterial cellulose in dimethyl sulfoxide at a ratio of 1g:(16-20)mL, then add the above activated oleic acid solution, and stir the reaction at 60-70℃ for 24-36h under nitrogen protection. After alcohol precipitation, centrifugation, washing and freeze drying, a composite nanocarrier is obtained, wherein the mass ratio of bacterial cellulose to oleic acid is 1:(2-2.2).

[0033] Furthermore, S4 specifically includes the following steps:

[0034] S4.1: Dissolve the composite nanocarrier obtained in step S3.4 in dimethyl sulfoxide at a ratio of 1g:(20-30)mL to obtain a composite nanocarrier solution. Then add verbena extract solution and moringa leaf extract solution, stir and mix evenly to obtain a mixed solution.

[0035] S4.2: Dissolve Tween 80 in chloroform at a ratio of 1g:(40-50)mL, then add the above mixed solution which accounts for 3 / 5 of the volume of chloroform, and homogenize and emulsify at 2000-3000rpm for 10-20min to obtain a mixed emulsion.

[0036] S4.3: The above mixed emulsion was rotary evaporated to remove chloroform, then filtered through a 0.45μm membrane, centrifuged, washed and freeze-dried to obtain the extract composite additive.

[0037] Furthermore, the mass ratio of acetic anhydride to resistant starch is (1.6-1.8):1, and the mass ratio of concanavalin A to resistant starch acetate is 1:(95-105).

[0038] Furthermore, the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:2, and the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to oleic acid is 1:(3.6-4).

[0039] Furthermore, the volume ratios of verbena extract solution and moringa leaf extract solution to composite nanocarrier solution were 1:(3-4) and 1:(4-6), respectively, and the concentrations of verbena extract solution and moringa leaf extract solution were both 50 mg / mL.

[0040] Furthermore, by weight, the raw material composition of the compound feed is as follows: 40-50 parts corn, 10-20 parts broken rice, 10-20 parts soybean meal, 10-15 parts eggshell powder, 10-20 parts spirulina powder, 8-14 parts xylooligosaccharides, 8-10 parts extract compound additives, 3-5 parts bifidobacteria compound inoculant and 2-4 parts compound vitamins, wherein the compound vitamins are composed of vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin D and vitamin E in a weight ratio of (1-2):(3-5):(3-4):1:(2-3):(1-1.2):(1.3-1.5).

[0041] Furthermore, a compound feed for improving the disease resistance of breeding ducks is prepared by the preparation process of a compound feed for improving the disease resistance of breeding ducks described in any of the above claims.

[0042] Compared with the prior art, the present invention has at least the following beneficial effects:

[0043] 1. In this invention, resistant starch acetate is activated with 1,1-carbonyldiimidazole, and then a solution of concanavalin A is added to react and form an amide bond. This allows concanavalin A to be grafted into the resistant starch acetate molecule, resulting in modified resistant starch acetate. When used to load Bifidobacteria, it can protect Bifidobacteria from damage by gastric acid and digestive enzymes, thereby increasing the number of Bifidobacteria reaching the large intestine of breeding ducks. This is beneficial for better inhibiting the reproduction of harmful bacteria in the large intestine of breeding ducks, reducing the risk of intestinal inflammation, and thus enhancing the immunity and disease resistance of breeding ducks. In addition, after modification with resistant starch acetate, concanavalin A can form a specific binding with the surface of Bifidobacteria through sugar-protein interactions, thereby reducing the shedding of Bifidobacteria during the loading process and helping to improve the loading rate of Bifidobacteria by resistant starch acetate.

[0044] 2. In this invention, bacterial cellulose is first hydrolyzed and acidified using sulfuric acid, and oleic acid is activated. Then, the acidified bacterial cellulose reacts with the activated oleic acid to form covalent bonds, thus creating a composite nanocarrier. After loading verbena extract and moringa leaf extract onto this composite nanocarrier, the introduction of oleic acid molecules increases the specific surface area and expands the pore capacity of the nanocarrier, enabling it to accommodate more extract molecules. This effectively improves the encapsulation rate of verbena extract and moringa leaf extract on the composite nanocarrier, which is beneficial for improving the disease-resistant efficacy of the extract composite additive, thereby enhancing the disease resistance of breeding ducks.

[0045] 3. In this invention, on the one hand, verbena extract can neutralize reactive oxygen species and reduce oxidative damage to immune cells, while the active antioxidant components in moringa leaf extract can further scavenge free radicals. The combined use of the two can significantly enhance the body's antioxidant capacity, reduce immune cell apoptosis, and maintain the efficient operation of the immune system. On the other hand, verbena extract can enhance immune cell activity by regulating cytokines, while moringa leaf extract can promote lymphocyte proliferation and differentiation, and enhance cellular immunity. The combined use of the two can significantly increase immunoglobulin levels, enhance the synergistic effect of humoral immunity and cellular immunity, and improve the clearance efficiency of pathogenic microorganisms. Therefore, verbena extract and moringa leaf extract can synergistically enhance the immunity of breeding ducks, thereby improving their disease resistance. Attached Figure Description

[0046] The accompanying drawings, which are incorporated herein and form part of the specification, illustrate embodiments of the present disclosure and, together with the specification, further serve to explain the principles of the present disclosure and enable those skilled in the art to implement and use the present disclosure.

[0047] Figure 1 This is a flowchart illustrating the preparation process of the compound feed used in this embodiment of the invention to improve the disease resistance of breeding ducks. Detailed Implementation

[0048] The following describes in detail, with reference to the accompanying drawings and specific embodiments, a compound feed for improving the disease resistance of breeding ducks and its preparation process.

[0049] Example 1

[0050] A process for preparing a compound feed to improve the disease resistance of breeding ducks, such as... Figure 1 As shown, it includes the following steps:

[0051] S1: Preparation of modified resistant starch acetate

[0052] S1.1: Add resistant starch to deionized water at a ratio of 1g:10mL, stir thoroughly to form a suspension, and cool in an ice bath to 3°C to obtain a resistant starch suspension;

[0053] S1.2: Add 0.1 mol / L sodium hydroxide solution to the above resistant starch suspension to adjust the pH to 8, and stir to swell for 20 min. Then add acetic anhydride, stir and react at 20℃ for 2 h, then add 0.1 mol / L hydrochloric acid solution to adjust the pH to neutral. Then collect the precipitate by centrifugation, wash and vacuum dry to obtain resistant starch acetate, wherein the mass ratio of acetic anhydride to resistant starch is 1.6:1.

[0054] S1.3: The above resistant starch acetate was suspended in acetone at a ratio of 1g:20mL, and 1,1-carbonyldiimidazole was added. The mixture was stirred at 25°C for 10h to obtain an activated resistant starch acetate solution, wherein the mass ratio of 1,1-carbonyldiimidazole to resistant starch acetate was 1:4.

[0055] S1.4: Dissolve canavalia protein A in PBS buffer at a ratio of 1g:150mL, then add it to the above activated resistant starch acetate solution, stir at 3℃ for 12h, then dialysis for purification and freeze-drying to obtain modified resistant starch acetate, wherein the mass ratio of canavalia protein A to resistant starch acetate is 1:95;

[0056] S2: Preparation of Bifidobacterium compound inoculum

[0057] S2.1: Inoculate the lyophilized Bifidobacterium powder into MRS medium, anaerobically culture at 37°C for 40 h, centrifuge at 4°C, discard the supernatant, wash twice with sterile physiological saline, and then resuspend in sterile PBS buffer to obtain a concentration of 10. 10 Bifidobacterium suspension with CUF / mL;

[0058] S2.2: Dissolve the modified resistant starch acetate obtained in step S1.4 in sterile PBS buffer at a ratio of 1g:20mL, then add the above-mentioned Bifidobacterium suspension, stir and mix thoroughly to obtain an aqueous phase, wherein the concentration of Bifidobacterium in the aqueous phase is approximately 4×10⁻⁶. 9 CUF / mL;

[0059] S2.3: Add Span 80 to liquid paraffin at a ratio of 1g:40mL, stir to dissolve, add the above aqueous phase, and homogenize and emulsify at 1500rpm for 10min to obtain an emulsion, wherein the volume ratio of aqueous phase to liquid paraffin is 1:2.

[0060] S2.4: Add 0.1 mol / L calcium chloride solution to the above emulsion, heat and stir at 37°C for 20 min for cross-linking, then centrifuge at 3°C, discard the supernatant, wash with sterile petroleum ether and sterile PBS buffer, and freeze dry to obtain Bifidobacterium complex agent, wherein the volume ratio of calcium chloride solution to water phase is 1:6.

[0061] S3: Preparation of composite nanocarriers

[0062] S3.1: Add bacterial cellulose to phosphate buffer at pH 5 at a ratio of 1g:20mL, and sonicate for 30min to obtain a bacterial cellulose suspension;

[0063] S3.2: Add a 64% sulfuric acid solution to the above bacterial cellulose suspension, stir and hydrolyze at 40°C for 14 hours, then add 1 mol / L sodium hydroxide solution to adjust the pH to neutral, and obtain acidified bacterial cellulose by centrifugation, washing and freeze drying, wherein the volume ratio of sulfuric acid solution to bacterial cellulose suspension is 1:2.

[0064] S3.3: Add oleic acid to chloroform at a volume ratio of 1:8 mL, stir thoroughly, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir in the dark for 1 h to activate, and obtain an activated oleic acid solution, wherein the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:2, and the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to oleic acid is 1:3.6;

[0065] S3.4: The above acidified bacterial cellulose was dispersed in dimethyl sulfoxide at a ratio of 1g:16mL, and the above activated oleic acid solution was added. Under nitrogen protection, the mixture was stirred at 60°C for 24h. After alcohol precipitation, centrifugation, washing and freeze drying, a composite nanocarrier was obtained, wherein the mass ratio of bacterial cellulose to oleic acid was 1:2.

[0066] S4: Preparation of extract composite additives

[0067] S4.1: Dissolve the composite nanocarrier obtained in step S3.4 in dimethyl sulfoxide at a ratio of 1g:20mL to obtain a composite nanocarrier solution. Then add verbena extract solution and moringa leaf extract solution, stir and mix evenly to obtain a mixed solution. The volume ratios of verbena extract solution and moringa leaf extract solution to composite nanocarrier solution are 1:3 and 1:4, respectively, and the concentrations of verbena extract solution and moringa leaf extract solution are both 50mg / mL.

[0068] The method for preparing verbena extract solution is as follows: Verbena leaves are crushed at a ratio of 1g:20mL and added to 70% ethanol solution. The mixture is heated to reflux at 70℃ for 2-3 hours and extracted. This process is repeated twice. The extracts are then combined and concentrated under reduced pressure until no ethanol remains. The mixture is extracted twice with petroleum ether and three times with ethyl acetate. The ethyl acetate phases are combined and evaporated to dryness under reduced pressure to obtain verbena extract. The verbena extract is then dissolved in 50% ethanol solution to obtain verbena extract solution.

[0069] The preparation method of Moringa leaf extract solution is as follows: Moringa leaf powder is added to 70% ethanol solution at a ratio of 1g:10mL, ultrasonically treated at 300W and 40℃ for 30min, the supernatant is collected by centrifugation, concentrated under reduced pressure to a paste, then reconstituted with deionized water, and freeze-dried to obtain Moringa leaf extract. Finally, Moringa leaf extract is dissolved in 50% ethanol solution to obtain Moringa leaf extract.

[0070] S4.2: Dissolve Tween 80 in chloroform at a ratio of 1g:40mL, then add the above mixed solution which accounts for 3 / 5 of the volume of chloroform, and homogenize and emulsify at 2000rpm for 10min to obtain a mixed emulsion;

[0071] S4.3: The above mixed emulsion was rotary evaporated to remove chloroform, then filtered through a 0.45μm membrane, centrifuged, washed and freeze-dried to obtain the extract composite additive;

[0072] S5: Preparation of compound feed

[0073] After mixing and pulverizing corn, broken rice, soybean meal, and eggshell powder, the above-mentioned extract compound additive, bifidobacteria compound inoculant, xylooligosaccharide, compound microorganisms, and spirulina powder are added. The mixture is thoroughly mixed and granulated to obtain a compound feed. The raw material composition of the compound feed, by weight, is: 40 parts corn, 10 parts broken rice, 10 parts soybean meal, 10 parts eggshell powder, 10 parts spirulina powder, 8 parts xylooligosaccharide, 8 parts extract compound additive, 3 parts bifidobacteria compound inoculant, and 2 parts compound vitamins. The compound vitamins consist of vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin D, and vitamin E in a weight ratio of 1:3:3:1:2:1:1.3.

[0074] Example 2

[0075] A process for preparing a compound feed to improve the disease resistance of breeding ducks, such as... Figure 1 As shown, it includes the following steps:

[0076] S1: Preparation of modified resistant starch acetate

[0077] S1.1: Add resistant starch to deionized water at a ratio of 1g:15mL, stir thoroughly to form a suspension, and cool in an ice bath to 4°C to obtain a resistant starch suspension;

[0078] S1.2: Add 0.1 mol / L sodium hydroxide solution to the above resistant starch suspension to adjust the pH to 9, and stir to swell for 25 min. Then add acetic anhydride, stir and react at 25 °C for 2.5 h, then add 0.1 mol / L hydrochloric acid solution to adjust the pH to neutral. Then collect the precipitate by centrifugation, wash and vacuum dry to obtain resistant starch acetate, wherein the mass ratio of acetic anhydride to resistant starch is 1.7:1.

[0079] S1.3: The above resistant starch acetate was suspended in acetone at a ratio of 1g:25mL, and 1,1-carbonyldiimidazole was added. The mixture was stirred at 30°C for 11h to obtain an activated resistant starch acetate solution, wherein the mass ratio of 1,1-carbonyldiimidazole to resistant starch acetate was 1:4.5.

[0080] S1.4: Dissolve canavalia protein A in PBS buffer at a ratio of 1g:175mL, then add it to the above activated resistant starch acetate solution, stir at 4℃ for 14h, then dialysis for purification and freeze-drying to obtain modified resistant starch acetate, wherein the mass ratio of canavalia protein A to resistant starch acetate is 1:100.

[0081] S2: Preparation of Bifidobacterium compound inoculum

[0082] S2.1: Inoculate the lyophilized Bifidobacterium powder into MRS medium, anaerobically culture at 37°C for 44 h, centrifuge at 4°C, discard the supernatant, wash twice with sterile physiological saline, and then resuspend in sterile PBS buffer to obtain a concentration of 10. 10 Bifidobacterium suspension with CUF / mL;

[0083] S2.2: Dissolve the modified resistant starch acetate obtained in step S1.4 in sterile PBS buffer at a ratio of 1g:25mL, then add the above-mentioned Bifidobacterium suspension, stir and mix thoroughly to obtain an aqueous phase, wherein the concentration of Bifidobacterium in the aqueous phase is approximately 4.5×10⁻⁶. 9 CUF / mL;

[0084] S2.3: Add Span 80 to liquid paraffin at a ratio of 1g:45mL, stir to dissolve, add the above aqueous phase, and homogenize and emulsify at 2000rpm for 15min to obtain an emulsion, wherein the volume ratio of aqueous phase to liquid paraffin is 1:2.

[0085] S2.4: Add 0.1 mol / L calcium chloride solution to the above emulsion, heat and stir at 37°C for 25 min for cross-linking, then centrifuge at 4°C, discard the supernatant, wash with sterile petroleum ether and sterile PBS buffer, and freeze dry to obtain Bifidobacterium complex agent, wherein the volume ratio of calcium chloride solution to water phase is 1:7.

[0086] S3: Preparation of composite nanocarriers

[0087] S3.1: Add bacterial cellulose to phosphate buffer solution with pH 5.5 at a ratio of 1g:25mL, and sonicate for 35min to obtain bacterial cellulose suspension;

[0088] S3.2: Add a 64% sulfuric acid solution to the above bacterial cellulose suspension, stir and hydrolyze at 45°C for 15 hours, then add a 1 mol / L sodium hydroxide solution to adjust the pH to neutral, and obtain acidified bacterial cellulose by centrifugation, washing and freeze drying. The volume ratio of sulfuric acid solution to bacterial cellulose suspension is 1:2.5.

[0089] S3.3: Add oleic acid to chloroform at a volume ratio of 1:9 mL, stir thoroughly, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir in the dark for 1.5 h to activate, and obtain an activated oleic acid solution, wherein the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:2, and the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to oleic acid is 1:3.8;

[0090] S3.4: The above acidified bacterial cellulose was dispersed in dimethyl sulfoxide at a ratio of 1g:18mL, and the above activated oleic acid solution was added. The mixture was stirred at 65°C for 30h under nitrogen protection. After alcohol precipitation, centrifugation, washing and freeze drying, a composite nanocarrier was obtained, wherein the mass ratio of bacterial cellulose to oleic acid was 1:2.1.

[0091] S4: Preparation of extract composite additives

[0092] S4.1: Dissolve the composite nanocarrier obtained in step S3.4 in dimethyl sulfoxide at a ratio of 1g:25mL to obtain a composite nanocarrier solution. Then add verbena extract solution and moringa leaf extract solution, stir and mix evenly to obtain a mixed solution. The volume ratios of verbena extract solution and moringa leaf extract solution to composite nanocarrier solution are 1:3.5 and 1:5, respectively, and the concentrations of verbena extract solution and moringa leaf extract solution are both 50mg / mL.

[0093] The method for preparing verbena extract solution is as follows: Verbena leaves are crushed at a ratio of 1g:25mL and added to 70% ethanol solution. The mixture is heated to reflux at 75℃ for 2.5h and extracted twice. After repeating the extraction twice, the extracts are combined and concentrated under reduced pressure until no ethanol remains. The mixture is then extracted twice with petroleum ether and three times with ethyl acetate. The ethyl acetate phases are combined and evaporated to dryness under reduced pressure to obtain verbena extract. The verbena extract is then dissolved in 50% ethanol solution to obtain verbena extract solution.

[0094] The preparation method of Moringa leaf extract solution is as follows: Moringa leaf powder is added to 70% ethanol solution at a ratio of 1g:15mL, ultrasonically treated at 350W and 45℃ for 35min, the supernatant is collected by centrifugation, concentrated under reduced pressure to a paste, then reconstituted with deionized water, and freeze-dried to obtain Moringa leaf extract. Finally, Moringa leaf extract is dissolved in 50% ethanol solution to obtain Moringa leaf extract.

[0095] S4.2: Dissolve Tween 80 in chloroform at a ratio of 1g:45mL, then add the above mixed solution which accounts for 3 / 5 of the volume of chloroform, and homogenize and emulsify at 2500rpm for 15min to obtain a mixed emulsion;

[0096] S4.3: The above mixed emulsion was rotary evaporated to remove chloroform, then filtered through a 0.45μm membrane, centrifuged, washed and freeze-dried to obtain the extract composite additive;

[0097] S5: Preparation of compound feed

[0098] Corn, broken rice, soybean meal, and eggshell powder are mixed and pulverized. Then, the above-mentioned extract compound additive, bifidobacteria compound inoculant, xylooligosaccharide, compound microorganisms, and spirulina powder are added, thoroughly mixed, and granulated to obtain compound feed. The raw material composition of the compound feed, by weight, is: 45 parts corn, 15 parts broken rice, 15 parts soybean meal, 12 parts eggshell powder, 15 parts spirulina powder, 11 parts xylooligosaccharide, 9 parts extract compound additive, 4 parts bifidobacteria compound inoculant, and 3 parts compound vitamins. The compound vitamins consist of vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin D, and vitamin E in a weight ratio of 1.5:4:3.5:1:2.5:1.1:1.4.

[0099] Example 3

[0100] A process for preparing a compound feed to improve the disease resistance of breeding ducks, such as... Figure 1 As shown, it includes the following steps:

[0101] S1: Preparation of modified resistant starch acetate

[0102] S1.1: Add resistant starch to deionized water at a ratio of 1g:20mL, stir thoroughly to form a suspension, and cool in an ice bath to 5°C to obtain a resistant starch suspension;

[0103] S1.2: Add 0.1 mol / L sodium hydroxide solution to the above resistant starch suspension to adjust the pH to 10, and stir to swell for 30 min. Then add acetic anhydride, stir and react at 30℃ for 3 h, then add 0.1 mol / L hydrochloric acid solution to adjust the pH to neutral. Then collect the precipitate by centrifugation, wash and vacuum dry to obtain resistant starch acetate, wherein the mass ratio of acetic anhydride to resistant starch is 1.8:1.

[0104] S1.3: The above resistant starch acetate was suspended in acetone at a ratio of 1g:30mL, and 1,1-carbonyldiimidazole was added. The mixture was stirred at 35°C for 12h to obtain an activated resistant starch acetate solution, wherein the mass ratio of 1,1-carbonyldiimidazole to resistant starch acetate was 1:5.

[0105] S1.4: Dissolve canavalia protein A in PBS buffer at a ratio of 1g:200mL, then add it to the above activated resistant starch acetate solution, stir at 5℃ for 16h, then dialysis and freeze dry to obtain modified resistant starch acetate, wherein the mass ratio of canavalia protein A to resistant starch acetate is 1:105.

[0106] S2: Preparation of Bifidobacterium compound inoculum

[0107] S2.1: Inoculate the lyophilized Bifidobacterium powder into MRS medium, anaerobically culture at 37°C for 48 h, centrifuge at 4°C, discard the supernatant, wash three times with sterile physiological saline, and then resuspend in sterile PBS buffer to obtain a concentration of 10. 10 Bifidobacterium suspension with CUF / mL;

[0108] S2.2: Dissolve the modified resistant starch acetate obtained in step S1.4 in sterile PBS buffer at a ratio of 1g:30mL, then add the above-mentioned Bifidobacterium suspension, stir and mix thoroughly to obtain an aqueous phase, wherein the concentration of Bifidobacterium in the aqueous phase is approximately 5×10⁻⁶. 9 CUF / mL;

[0109] S2.3: Add Span 80 to liquid paraffin at a ratio of 1g:50mL, stir to dissolve, add the above aqueous phase, and homogenize and emulsify at 2500rpm for 20min to obtain an emulsion, wherein the volume ratio of aqueous phase to liquid paraffin is 1:2.

[0110] S2.4: Add 0.1 mol / L calcium chloride solution to the above emulsion, heat and stir at 37°C for 30 min for cross-linking, then centrifuge at 5°C, discard the supernatant, wash with sterile petroleum ether and sterile PBS buffer, and freeze dry to obtain Bifidobacterium complex agent, wherein the volume ratio of calcium chloride solution to water phase is 1:8;

[0111] S3: Preparation of composite nanocarriers

[0112] S3.1: Add bacterial cellulose to phosphate buffer at pH 6 at a ratio of 1g:30mL, and sonicate for 40min to obtain bacterial cellulose suspension;

[0113] S3.2: Add a 64% sulfuric acid solution to the above bacterial cellulose suspension, stir and hydrolyze at 50°C for 16 hours, then add a 1 mol / L sodium hydroxide solution to adjust the pH to neutral, and obtain acidified bacterial cellulose by centrifugation, washing and freeze drying, wherein the volume ratio of sulfuric acid solution to bacterial cellulose suspension is 1:3.

[0114] S3.3: Add oleic acid to chloroform at a volume ratio of 1:10 mL, stir thoroughly, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir and activate in the dark for 2 h to obtain an activated oleic acid solution, wherein the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:2, and the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to oleic acid is 1:4;

[0115] S3.4: The above acidified bacterial cellulose was dispersed in dimethyl sulfoxide at a ratio of 1g:20mL, and then the above activated oleic acid solution was added. Under nitrogen protection, the mixture was stirred at 70°C for 36h. After alcohol precipitation, centrifugation, washing and freeze drying, a composite nanocarrier was obtained, wherein the mass ratio of bacterial cellulose to oleic acid was 1:2.2.

[0116] S4: Preparation of extract composite additives

[0117] S4.1: Dissolve the composite nanocarrier obtained in step S3.4 in dimethyl sulfoxide at a ratio of 1g:30mL to obtain a composite nanocarrier solution. Then add verbena extract solution and moringa leaf extract solution, stir and mix evenly to obtain a mixed solution. The volume ratios of verbena extract solution and moringa leaf extract solution to composite nanocarrier solution are 1:4 and 1:6, respectively, and the concentrations of verbena extract solution and moringa leaf extract solution are both 50mg / mL.

[0118] The method for preparing verbena extract solution is as follows: Verbena leaves are crushed at a ratio of 1g:30mL and added to 70% ethanol solution. The mixture is heated to reflux at 80℃ for 3h and extracted. This process is repeated twice. The extracts are then combined and concentrated under reduced pressure until no ethanol remains. The mixture is extracted twice with petroleum ether and three times with ethyl acetate. The ethyl acetate phases are combined and evaporated to dryness under reduced pressure to obtain verbena extract. The verbena extract is then dissolved in 50% ethanol solution to obtain verbena extract solution.

[0119] The preparation method of Moringa leaf extract solution is as follows: Moringa leaf powder is added to 70% ethanol solution at a ratio of 1g:20mL, ultrasonically treated at 400W and 50℃ for 40min, the supernatant is collected by centrifugation, concentrated under reduced pressure to a paste, then reconstituted with deionized water, and freeze-dried to obtain Moringa leaf extract. Finally, Moringa leaf extract is dissolved in 50% ethanol solution to obtain Moringa leaf extract.

[0120] S4.2: Dissolve Tween 80 in chloroform at a ratio of 1g:50mL, then add the above mixed solution which accounts for 3 / 5 of the volume of chloroform, and homogenize and emulsify at 3000rpm for 20min to obtain a mixed emulsion;

[0121] S4.3: The above mixed emulsion was rotary evaporated to remove chloroform, then filtered through a 0.45μm membrane, centrifuged, washed and freeze-dried to obtain the extract composite additive;

[0122] S5: Preparation of compound feed

[0123] After mixing and pulverizing corn, broken rice, soybean meal, and eggshell powder, the above-mentioned extract compound additive, bifidobacteria compound inoculant, xylooligosaccharide, compound microorganisms, and spirulina powder are added. The mixture is thoroughly mixed and granulated to obtain a compound feed. The raw material composition of the compound feed, by weight, is: 50 parts corn, 20 parts broken rice, 20 parts soybean meal, 15 parts eggshell powder, 20 parts spirulina powder, 14 parts xylooligosaccharide, 10 parts extract compound additive, 5 parts bifidobacteria compound inoculant, and 4 parts compound vitamins. The compound vitamins consist of vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin D, and vitamin E in a weight ratio of 2:5:4:1:3:1.2:1.5.

[0124] Comparative Example 1

[0125] The difference between Comparative Example 1 and Example 1 is that step S1 is removed, and the modified resistant starch acetate in step S2.2 is replaced with an equal amount of resistant starch acetate.

[0126] Comparative Example 2

[0127] The difference between Comparative Example 2 and Example 1 is that step S1 is removed, and the modified resistant starch acetate in step S2.2 is replaced with an equal amount of resistant starch.

[0128] Comparative Example 3

[0129] The difference between Comparative Example 3 and Example 1 is that step S3 is removed, and the composite nanocarrier in step S4 is replaced with an equal amount of acidified bacterial cellulose.

[0130] Comparative Example 4

[0131] The difference between Comparative Example 4 and Example 1 is that the Moringa leaf extract solution in step S4 is replaced with an equal amount of Verbena officinalis extract solution.

[0132] Comparative Example 5

[0133] The difference between Comparative Example 5 and Example 1 is that the verbena extract solution in step S4 is replaced with an equal amount of moringa leaf extract solution.

[0134] Test case

[0135] Test 1: The loading rate of Bifidobacterium in the Bifidobacterium compound inoculants prepared in Examples 1-3 and Comparative Examples 1-2 was tested, and the results are shown in Table 1.

[0136] Table 1: Results of Bifidobacterium loading rate test

[0137]

[0138] As shown in Table 1, when Bifidobacteria were loaded directly with resistant starch acetate or resistant starch in Comparative Examples 1 and 2, the loading rate of Bifidobacteria was lower than that in Example 1. This shows that esterification of resistant starch and modification of resistant starch acetate with concanavalin A can improve the loading rate of resistant starch on Bifidobacteria, which is beneficial to better inhibit the reproduction of harmful Escherichia coli in breeding ducks, reduce the risk of intestinal inflammation, and thus enhance the immunity of breeding ducks and improve their disease resistance.

[0139] Test 2: The encapsulation efficiency of the total amount of verbena extract and moringa leaf extract in the extract composite additives prepared in Examples 1-3 and Comparative Example 3 was tested, and the results are shown in Table 2.

[0140] Table 2: Encapsulation efficiency test results of total amount of verbena extract and moringa leaf extract

[0141]

[0142] As shown in Table 2, in Comparative Example 3, without reacting with oleic acid, the encapsulation rate of verbena extract and moringa leaf extract was much lower than that in Example 1 after directly loading the acidified bacterial cellulose with the oleic acid. This indicates that by first hydrolyzing and acidifying the bacterial cellulose with sulfuric acid and activating the oleic acid, and then reacting the acidified bacterial cellulose with the activated oleic acid to form covalent bonds and prepare a composite nanocarrier, and then using this composite nanocarrier to load verbena extract and moringa leaf extract, the encapsulation rate of verbena extract and moringa leaf extract can be effectively improved. This is beneficial to improving the disease-resistant efficacy of the extract composite additive, thereby enhancing the disease resistance of breeding ducks.

[0143] Test 3: 600 white-feathered parent ducks were randomly selected and divided into 6 groups of 100 each. Each group was fed the compound feed prepared in Examples 1-3 and Comparative Examples 4-5 according to the conventional breeding process of the farm. The remaining group was a blank group, which was fed only conventional commercial basic feed. After 4 weeks of feeding, 10 ducks were randomly selected from each group to collect blood from the wing vein. 5 mL of blood was collected from each duck and then centrifuged at 3000 r / min for 15 min. The serum immunoglobulin (IgG, IgM, IgA) index was measured by ELISA and recorded and analyzed. The average value was taken. The results are shown in Table 3.

[0144] Table 3: Results of Immunoglobulin Index Tests

[0145]

[0146] As shown in Table 3, when only one of verbena extract and moringa leaf extract was used in Comparative Examples 4 and 5, the immune indicators of the breeding ducks fed with the compound feed were higher than those of the blank group, but lower than those of Example 1. This shows that verbena extract and moringa leaf extract can synergistically enhance the immunity of breeding ducks, thereby improving their disease resistance.

[0147] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A process for preparing a compound feed to improve the disease resistance of breeding ducks, characterized in that, Includes the following steps: S1: Preparation of modified resistant starch acetate, S1.1: Add resistant starch to deionized water to form a suspension, and cool it in an ice bath to 3-5℃ to obtain a resistant starch suspension; S1.2: Add sodium hydroxide solution to the above resistant starch suspension to adjust the pH to 8-10, then add acetic anhydride, stir to react, and then adjust the pH to neutral to obtain resistant starch acetate. S1.3: Suspend resistant starch acetate in acetone and add 1,1-carbonyldiimidazole. Stir and activate at 25-35℃ for 10-12 h to obtain an activated resistant starch acetate solution. S1.4: Dissolve canavon protein A in PBS buffer, then add it to the activated resistant starch acetate solution, stir at 3-5℃ for 12-16 h, then purify by dialysis and freeze dry to obtain modified resistant starch acetate; The mass ratio of acetic anhydride to resistant starch is (1.6-1.8):1, and the mass ratio of canavalia protein A to resistant starch acetate is 1:(95-105). S2: Preparation of Bifidobacterium compound inoculum Bifidobacterium lyophilized powder was activated and then mixed with modified resistant starch acetate solution to prepare a compound inoculum of Bifidobacterium; S3: Preparation of composite nanocarriers S3.1: Add bacterial cellulose to phosphate buffer solution with pH 5-6 at a ratio of 1g:(20-30)mL, and sonicate for 30-40min to obtain bacterial cellulose suspension; S3.2: Add sulfuric acid solution to the above bacterial cellulose suspension and stir to hydrolyze, then adjust the pH to neutral to prepare acidified bacterial cellulose, wherein the volume ratio of sulfuric acid solution to bacterial cellulose suspension is 1:(2-3). S3.3: Add oleic acid to chloroform at a volume ratio of 1:(8-10) mL, stir and mix thoroughly, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide at a mass ratio of 1:2, stir and activate in the dark for 1-2 h to obtain an activated oleic acid solution; S3.4: Acidified bacterial cellulose was dispersed in dimethyl sulfoxide, and then activated oleic acid solution was added. The mixture was stirred at 60-70℃ for 24-36 h under nitrogen protection. After alcohol precipitation, centrifugation, washing and freeze drying, a composite nanocarrier was obtained, wherein the mass ratio of bacterial cellulose to oleic acid was 1:(2-2.2). S4: Preparation of extract compound additives S4.1: Dissolve the composite nanocarrier obtained in step S3.4 in dimethyl sulfoxide at a ratio of 1g:(20-30)mL to obtain a composite nanocarrier solution. Then add verbena extract solution and moringa leaf extract solution, stir and mix evenly to obtain a mixed solution. S4.2: Dissolve Tween 80 in chloroform at a ratio of 1g:(40-50)mL, then add the above mixed solution which accounts for 3 / 5 of the volume of chloroform, and homogenize and emulsify for 10-20 minutes to obtain a mixed emulsion; S4.3: The mixed emulsion is rotary evaporated to remove chloroform, then filtered through a membrane, centrifuged, washed and freeze-dried to obtain the extract composite additive; The volume ratio of verbena extract solution to composite nanocarrier solution is 1:(3-4), and the volume ratio of moringa leaf extract solution to composite nanocarrier solution is 1:(4-6), and the concentration of verbena extract solution and moringa leaf extract solution is 50mg / mL. S5: Prepare compound feed. After mixing and crushing corn, broken rice, soybean meal and eggshell powder, add the above-mentioned extract compound additive, bifidobacteria compound inoculant, xylooligosaccharide, compound microorganisms and spirulina powder, mix thoroughly and granulate to obtain compound feed.

2. The preparation process of a compound feed for improving the disease resistance of breeding ducks according to claim 1, characterized in that, S2 specifically includes the following steps: S2.1: Inoculate the lyophilized Bifidobacterium powder into MRS medium, anaerobically incubate at 37°C for 40-48 h, centrifuge at 4°C, discard the supernatant, wash and resuspend with sterile physiological saline to obtain a concentration of 10. 10 A CUF / mL suspension of Bifidobacterium; S2.2: Dissolve the modified resistant starch acetate obtained in step S1.4, add the above-mentioned Bifidobacterium suspension, stir and mix evenly to obtain an aqueous phase, wherein the concentration of Bifidobacterium in the aqueous phase is (4-5)×10 9 CUF / mL; S2.3: Add Span 80 to liquid paraffin at a ratio of 1g:(40-50)mL, stir to dissolve, add the above aqueous phase, homogenize and emulsify for 10-20min to obtain an emulsion, wherein the volume ratio of aqueous phase to liquid paraffin is 1:2; S2.4: Add 0.1 mol / L calcium chloride solution to the above emulsion, heat and stir at 37°C for 20-30 min for cross-linking, then centrifuge, discard the supernatant, wash and freeze dry to obtain Bifidobacterium complex agent, wherein the volume ratio of calcium chloride solution to water phase is 1:(6-8).

3. The preparation process of a compound feed for improving the disease resistance of breeding ducks according to claim 1, characterized in that, The mass ratio of 1,1-carbonyldiimidazole to resistant starch acetate is 1:(4-5).

4. The preparation process of a compound feed for improving the disease resistance of breeding ducks according to claim 1, characterized in that, The mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to oleic acid is 1:(3.6-4).

5. The preparation process of a compound feed for improving the disease resistance of breeding ducks according to claim 1, characterized in that, By weight, the raw material composition of compound feed is as follows: 40-50 parts corn, 10-20 parts broken rice, 10-20 parts soybean meal, 10-15 parts eggshell powder, 10-20 parts spirulina powder, 8-14 parts xylooligosaccharides, 8-10 parts extract compound additives, 3-5 parts bifidobacteria compound inoculant and 2-4 parts compound vitamins.

6. A compound feed for improving the disease resistance of breeding ducks, characterized in that, It is prepared by the preparation process of a compound feed for improving the disease resistance of breeding ducks as described in any one of claims 1-5.

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